Composition and method for regulating the adhesion of cells and biomolecules to hydrophobic surfaces

ABSTRACT

The present invention is directed to a composition and method for regulating the adhesion of cells and biomolecules to hydrophobic surfaces and hydrophobic coated surfaces. The composition is a biomolecule conjugated end-group activated polymer (EGAP). The biomolecule conjugated EGAP can be put to numerous uses including cell adhesion, cell growth, cell sorting, and other biological assays.

1. RELATED APPLICATIONS

[0001] This application is a continuation-in-part of copending U.S.patent application Ser. No.08/399,913 filed Mar. 7, 1995, and entitled“Coating of Hydrophobic Surfaces to Render Them Protein Resistant WhilePermitting Covalent Bonding,” which is a divisional of U.S. Pat. No.5,516,703 issued May 14, 1996 and entitled “Coating of HydrophobicSurfaces to Render Them Protein Resistant While Permitting CovalentAttachment of Specific Ligands,” which applications are incorporatedherein by reference.

2. FIELD OF THE INVENTION

[0002] The present invention is related to a composition and method forregulating the adhesion of cells, organisms, and molecules tohydrophobic surfaces. More specifically, the present invention isdirected to a biomolecule, such as proteins, peptides amino acids,nucleic acids, lipids, and carbohydrates conjugated to end-groupactivated polymers (EGAPs) and uses thereof.

3. TECHNICAL BACKGROUND

[0003] Normal development and function in living organisms requireinteractions between cells and the molecules in the surroundingenvironment. One way cells communicate is via molecules that span themembrane of the cell called transmembrane proteins. When the portion ofthe transmembrane protein which is outside of the cell encountersspecific molecules in the surrounding environment, it undergoesstructural and conformational changes which triggers biologicalreactions inside the cell.

[0004] For example, in vivo, cells form complex multilayer structureswhich ultimately form tissues and organs. Tissue and organ formation,however, requires specific contacts with the environment. These cellsare referred to as “anchorage-dependent” because they will not growproperly, if at all, unless they are anchored to others cells, anextracellular matrix (ECM), or other surface.

[0005] An ECM is a complex and variable array of molecules secreted bycells, such as collagens, glycosaminoglycans, proteoglycans, andglycoproteins. Together these cellular products form the basal lamina,bone, and cartilage which give tissues and organs their shape andstrength. In fact, contact between anchorage-dependent cells and the ECMin many instances plays a dramatic role in determining the cells' shape,position, metabolism, differentiation and growth.

[0006] Cell contact is also important in other biological functions,such as the activation of an immune response. The immune system is acomplex network of cells that have the ability to recognize and rid thebody of foreign substances, such as viruses, bacteria and parasites. Onemechanism used by the immune system to rid itself of foreign substancesis a humoral response. A humoral response involves activation ofspecific cells called B cell lymphocytes. B-cells are activated whentransmembrane proteins on their surface bind to foreign substancescalled antigens. Specifically, binding of B-cells to antigens stimulatesB cells to proliferate and differentiate into immunoglobulin or antibodyproducing plasma cells.

[0007] The antibodies produced by plasma cells travel throughout thebody binding to the pathogen or foreign substance. Binding of antibodiesto foreign substances activates several other immunological pathways,including the “complement” pathway. The complement pathway is designedto destroy the foreign substance and to initiate an inflammatoryresponse in the organism.

[0008] While cell contact with other cells and the environment iscritical to the overall health and biological function of an organism,it creates unique problems in the art of biotechnology. Specifically,two areas where cell contact requirements create problems are: (1) cellculture; and (2) biomaterial transplantation.

[0009] Tissue or cell cultures comprise cells from a plant or animalwhich are grown outside the organism from which they originate. Thesecells are often grown, for example, in petri dishes under specificenvironmental conditions. Cell cultures are of great importance becausethey represent biological “factories” capable of producing largequantities of biological products such as growth factors, antibodies,and viruses. These products can then be isolated from the cell culturesand used, for example, to treat human disease. In addition, cellcultures are a potential source of tissue which could be used fortransplantation into humans. For example, cell cultured skin cells couldpotentially be used in skin grafts to replace diseased or damaged skin.Finally, cell cultures usually comprise cells from only one or a fewtissues or organs. Consequently, cell cultures provide scientists with asystem for studying the properties of individual cell types without thecomplications and risk of working with the entire organism. For example,the effects of pharmaceutical drugs on certain cell types could betested on cell cultures prior to clinical trials in order to assess thedrug's health risks.

[0010] Like most cells in vivo, cells grown in culture are eitheranchored to an ECM or another cell. Only cells of the circulatory system(e.g., lymphocytes and red blood cells) grow unattached and suspended insolution in vitro. Many anchorage-dependent cells can grow on glass orplastic surfaces, such as polystyrene. These cells, however, often losetheir natural architecture and do not function normally (e.g., theability to differentiate and respond to hormones). Accordingly, thesecells do not precisely mimic a cell's biological functions in vivo andthus have limited potential.

[0011] For this reason, glass and plastic cell culture dishes are oftencoated with an ECM protein such as collagen, fibronectin, laminin andthe like. These proteins bind to surfaces such as polystyrene through aprocess known as adsorption. Although ECM coated cell culture surfaceshave led to improved culture conditions, they are far from ideal.

[0012] First, biomolecules, such as proteins, often become inactivatedupon adsorption to hydrophobic surfaces. The biological activity ofproteins is conferred by their unique structure and their ability toundergo conformational changes upon binding to a substrate or otherphysiological event. In one study, the structure of proteins wasmeasured using a technique called microcalorimetry. Microcalorimetricstudies demonstrated that proteins which are bound to hydrophobicsurfaces loose essentially all their cooperatively folded structurecompared to the same protein in solution. Because a protein's structureand its ability to undergo conformational changes strongly correlateswith biological activity, these data suggest that most proteins that areadsorbed by a hydrophobic surface loose there in vivo biologicalactivity.

[0013] Second, the conformation and orientation of immobilized proteinshave important effects on the nature of their interaction with cells. D.J. Juliano, S. S. Saaedra and G. A. Truskey, Journal of BiomedicalMaterials Research 2-7 1103-1113 (1993). Both are influenced by thechemistry and physical properties of the underlying substrate as well asby the method of immobilization. K. Lewandowska, E. Pergament, N.Sukenik and L. A. Culp, The Journal of Biomedical Materials Research 211343-1363 (1992).

[0014] Third, like in vivo, cells in culture release molecules such asserum proteins and growth factors into the culture media. As discussedabove, the secretion and concentration of these molecules in the culturemedia are critical to the biological function of neighboring cells.Under current cell culture conditions, the careful balance andconcentration of secreted molecules are disrupted because secretedmolecules are adsorbed by the cell culture surface. Thus, thecommunication and biological function of cells grown under current cellculture techniques does not mimic in vivo environment.

[0015] Finally, the surface concentration of ECM components is acritical factor in the regulation of cell behavior. The ability tocontrol and vary surface biomolecule concentration is therefore ofupmost importance and depends on the method of immobilization and insome cases the physical nature of the base material. Simple ECMadsorption to cell culture substrates does not meet these requirements.

[0016] In short, to date there is no single method for conjugatingproteins to potential cell culture substrates which addresses all thesemajor concerns. Thus, current research is hindered by the fact that cellcultures do not accurately mimic an in vivo environment.

[0017] A second problem area created by cell contact isbiocompatibility. It is generally acknowledged that artificialbiomaterials, including fabricated biomedical polymers, are much lessimmunologically active than transplants or tissue-derived biomaterials.Nevertheless, the use of non-physiological biomaterials in manylifesaving medical devices, either extracorporeal or implanted, oftenleads to adverse side-effects for the patient.

[0018] The adverse side-effects observed are usually a consequence ofcontact between cells, proteins, and other biological fluids in theblood with the artificial biomaterial. Typically, contact with theartificial biomaterial activates two major biological processes:coagulation and complement. As discussed above, the complement pathwayis designed to destroy the foreign substance and to initiate aninflammatory response in the organism.

[0019] Activation of the coagulation cascade can be controlled to alimited extent with the use of anticoagulants, e.g., heparin. Heparin,however, is not well suited for extended use such as in the case of apermanent implant. Further, currently there is no clinically availableagent that can prevent or suppress artificial surface-initiatedactivation of complement. Thus, activation of the coagulation andcomplement systems upon blood contact is a major problem with respect tobiomaterial transplantation.

[0020] From the foregoing, it will be appreciated that it would be anadvancement in the art to provide a method of coating tissue culturesurfaces with ECM proteins or other biomolecules that does not destroythe biological activity of the biomolecule.

[0021] It would also be an advancement in the art if the biomoleculecoated surface could be used to adhere prokaryotic and eukaryotic cells,viruses, and other molecules for the purpose of biological assay.

[0022] It would be a further advancement in the art if the tissueculture cells could adhere and grow on the biomolecule coated surface.

[0023] It would be yet another advancement in the art if the biomoleculecoated surface did not adsorb proteins and other molecules secreted bythe cells in culture.

[0024] Finally, it would be an-advancement in the art if biomaterialused in transplantation could be coated with an immunologically inertbiomolecule to prevent or minimize host rejection.

[0025] Such compositions and methods are disclosed and claimed herein.

4. BRIEF SUMMARY OF THE INVENTION

[0026] The present invention is directed at a composition and method forregulating the adhesion of cells and biomolecules to hydrophobicsurfaces and hydrophobic coated surfaces. Generally, the composition isan end-group activated polymer (EGAP) generally comprises a blockcopolymer surfactant backbone and an activation or reactive group. Thepolymeric block copolymer surfactant of the present invention may be anysurfactant having a hydrophobic region capable of adsorbing onto ahydrophobic surface and a hydrophilic region which extends away from thesurface when the hydrophobic region is adsorbed to the hydrophobicsurface. In one embodiment, the EGAP is synthesized by reacting theblock copolymer surfactant with 4-nitrophenylchloroformate followed by2-(2-pyridyldithio)ethylamine.

[0027] A large range of biomolecules can be conjugated to EGAP, includenatural or recombinant growth factor, mitogens, growth peptides,differentiating factors, sugars, carbohydrates, polysaccharides, lipids,sterols, fatty acids and nucleic acid. In one embodiment, thebiomolecule contains a natural or artificial thiol group. Thesebiomolecules are conjugated to EGAP via a disulfide linkage.

[0028] The biomolecule conjugated EGAP surface can be put to a widevariety of uses. For example, the composition can be used to attachorganisms and molecules for growth or biological analysis. Briefly, thisis done by contacting a hydrophobic surface with an EGAP for a timesufficient for the EGAP to be adsorbed by the hydrophobic surface. Abiomolecule is then conjugated to the EGAP adsorbed to the hydrophobicsurface to form a biomolecule conjugated EGAP surface. After washing ofunconjugated biomolecule, organisms or molecules are placed in contactwith the biomolecule conjugated EGAP coated surface such that theorganism or molecule adheres to the biomolecule conjugated EGAP coatedsurface. In one embodiment, the organism or molecule is a eukaryotic orprokaryotic cell, a virus, an antibody or a pharmaceutical drug.

[0029] The biomolecule conjugated EGAP surface can also be used toselecting at least one desired organism or molecule from a mixture of atleast two organisms or molecules. This is done by first adsorbing EGAPonto a hydrophobic surface. A biomolecule unique for a desired organismor molecule being selected is then conjugated to the EGAP adsorbed tothe hydrophobic surface. A mixture of organisms or molecules containingthe desired organism or molecule is then contacted with the biomoleculeconjugated EGAP coated surface and the desired organism or molecule isallowed to adhere to the unique biomolecule. Finally, non-adheredorganisms or molecules are removed.

[0030] These and other objects and advantages of the present inventionwill become apparent upon reference to the accompanying drawings andgraphs and upon reading the following detailed description and appendedclaims.

5. SUMMARY OF THE DRAWINGS

[0031] A more particular descriptions of the invention briefly describedabove will be rendered by reference to the appended drawings and graphs.These drawings and graphs only provide information concerning typicalembodiments of the invention and are not therefore to be consideredlimiting of its scope.

[0032]FIG. 1 is a schematic representation of cells attached to a tissueculture surface coated with the composition of the present invention.

[0033]FIG. 2 is a graph illustrating the adhesiveness of NIH 3T3 cellsto Pluronic™ F108 coated hydrophobic surfaces.

[0034]FIG. 3 is a graph illustrating the thermal stability offibronectin (FN) adsorbed by a hydrophobic surface (PS261-FN), andconjugated to EGAP coated hydrophobic surface (PS261-EGAP-FN).Unconjugated EGAP adsorbed by a hydrophobic surface (PS261-EGAP) wasused as a control.

[0035]FIG. 4 is a graph illustrating the thermal stability of humanserum albumin (HSA) free in phosphate buffered saline solution (HSA inPBS), adsorbed by a hydrophobic surface (PS-HSA), and conjugated to EGAPcoated hydrophobic surface (PS-EGAP-HSA).

[0036]FIG. 5 is a picture of fibroblast cells attached and growing on afibronectin peptide GRGDSY conjugated EGAP coated surface.

[0037]FIG. 6 is a picture illustrating that fibroblast cells were unableto attach to unconjugated EGAP coated surface.

[0038]FIG. 7 is a graph illustrating the adhesion of cells to surfacescoated with F-108 (F108), F-108 containing unconjugated GRGDSY(F108/RGD), 2-pyridyl disulfide conjugated F-108 (PDSF108), GRGDSYconjugated EGAP (PDSF108/RGD), and untreated polystyrene (PS).

[0039]FIG. 8 is a picture illustrating that cells did not attach whenseeded on PEO modified surfaces but were found to attach, spread, andproliferate well on unmodified areas. FIG. 8a is a close-up displayingcells aligned at an interface between PEO treated and unmodified areas.In FIG. 8b, cells were fixed and removed from culture well afteradequate time to lay down a substantial ECM. A dark spot in the centercorresponds to the PEO treated area where there were no cells.

[0040]FIG. 9 illustrates that a hydrophobic surface coated withfibronectin peptide RGDS conjugated to EGAP (RGDS-PS) were found tosupport cell adhesion, (2-pyridyldithio)ethylamine modifiedEGAP(PDSF108-PS) displayed an intermediate level of adhesiveness, andF108 coated polystyrene was relatively non-adhesive to fibroblast cells.

6. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention is directed to a novel compound and methodfor regulating the adhesion of culture cells, organisms, and otherbiomolecules to a hydrophobic surface. More specifically, the inventionis directed to biomolecules that have been conjugated to end-groupactivated polymer (EGAPs). Biomolecule conjugated EGAPs lo can be usedto coat hydrophobic surfaces making them suitable for a wide range ofbiochemical and medical uses.

[0042] Reference is now made to FIG. 1. With reference to FIG. 1, in onepreferred embodiment, the present invention is a system 10 for attachingand growing cells in vitro. System 10 generally comprises a hydrophobictissue culture surface 20, biomolecule conjugated EGAP 30, and cells 60.

[0043] System 10 is constructed by first preparing a modified end-grouppolymer (EGAP) 40. In one embodiment, EGAP 40 comprises a hydrophobicblock 44 and two hydrophilic blocks 46. EGAP 40 is modified by, forexample, reacting at least one hydrophilic block 46 with 4-nitrophenylchloroformate followed by 2-(2-pyridyldithio) ethylamine.

[0044] EGAP 40 is then applied onto a hydrophobic tissue culture surface20. Upon application, hydrophobic block 44 of EGAP 40 is adsorbed byhydrophobic tissue culture surface 20. Hydrophilic blocks 46 do notadsorb, however. Instead, hydrophilic blocks 46 extend from the surfacein a “sea-weed” fashion. Once EGAP 30 has adsorbed onto the hydrophobictissue culture surface 20, excess EGAP 30 is removed and tissue culturesurface 20 is washed.

[0045] Simultaneously, biomolecule 50 is thiolated by methods well knownin the art. In one embodiment, biomolecule 50 is thiolated with reducedglutathione. EGAP 40 and biomolecule 50 are then reacted to formbiomolecule conjugated EGAP 30. After excess biomolecule conjugated EGAPhas been removed and tissue culture surface 20 has been washed, cells 60are seeded on the biomolecule conjugated EGAP coated surface 20. Cells60 attach to biomolecule 50, extend processes, and proliferate in anenvironment that resembles an in vivo setting.

[0046] In order to better understand the details of the presentinvention, the following discussion is divided in six sections: (1)hydrophobic surfaces; (2) EGAP; (3) binding EGAPs to hydrophobicsurfaces; (4) suitable biomolecule conjugates; (5) biomoleculeconjugated EGAPs; and (6) uses for biomolecule conjugated EGAP coatedsurfaces.

6.1 Hydrophobic Surfaces

[0047] The hydrophobic polymer surfaces of the present inventioncomprise any suitable polymer or surface coating material which impartsa hydrophobic character to the surface of the substrate. By“hydrophobic” is meant that the surface has a water contact anglegreater than about 60′, preferably greater than about 70′. Suitablepolymers or biomaterials with surfaces having a water contact anglegreater than 70′ include, but are not limited to polystyrene (PS),polymethylmethacrylate (PMMA), polyolefins (e.g. polyethylene (PE),polypropylene (PP)), polyvinylchloride (PVC), silicones,polyacrylonitrile (PAN), copolymers of polyacrylonitrile/polyvinalchloride, polysulfone, poly (ether sulfone) (PES), certainpolyurethanes, pyrolized materials, and block copolymers containingthese constituents.

[0048] Lesser hydrophobic polymer surfaces (water contact angles between60′ and 70′), such as PVAC are also contemplated by the invention butare less are preferred. Adsorption upon these polymers would be expectedto be reduced compared to more hydrophobic polymers such as PS and PMMA.Moreover, detachment of the block copolymer surfactant from the polymersurface over time would be expected. These and non-hydrophobic surfaces,however, may be treated to render them hydrophobic before blockcopolymer surfactant adsorption. For example, silica can be treated withdimethyldichloro silane to provide a hydrophobic surface.

[0049] The polymer may be porous or nonporous, or be in the form a flatsurface (e.g. a microliter plate), or any suitable shape, such as microbeads, and the like used in chromatography applications. The polymericsurfactant may also be adsorbed upon colloidal or latex particles of asuitable hydrophobic polymer.

6.2 End-Group Activated Polymers (EGAP)

[0050] As used herein, the terms end-group activated polymers (EGAP)refers to modified block copolymers surfactants. In one embodiment, theEGAPs of the present invention are of the type defined in U.S. Pat.No.5,516,703 entitled “Coating of Hydrophobic Surfaces to Render ThemProtein Resistant While Permitting Covalent Attachment of SpecificLigands” which is hereby incorporated by reference. Briefly, EGAP isblock copolymer surfactant where at least one of the hydrophilic chainshas been modified to make it chemically reactive to biomolecules.Accordingly, an EGAP generally comprises a block copolymer surfactantbackbone and an activation group or reactive group.

6.2.1 Block Copolymer Surfactant

[0051] The polymeric block copolymer surfactant of the present inventionmay be any surfactant having a hydrophobic region capable of adsorbingonto a hydrophobic surface and a hydrophilic region which extends awayfrom the surface when the hydrophobic region is adsorbed to thehydrophobic surface. In one embodiment, the block copolymer surfactantbackbone of EGAP may be in the form of any arrangement of the PEO andPPO blocks with the general formula:

(HO—PEO)_(a)(PPO)_(b)  (1)

[0052] where (a) and (b) are integers. Preferably (a) is between 1 and6, and (b) is between 1 and 3, more preferably (a) is 1 to 2, and (b)is 1. The polymeric block copolymer has a PEO (—C₂H₄—O—) content between10 wt % and 80 wt %, preferably 50 wt % and 80 wt %, and more preferablybetween 70 wt % and 80 wt %.

[0053] The PEO chains or blocks are of the general formula:

—(—C₂H₄—O—)_(u)—  (2)

[0054] where (u) is the same or different for different PEO blocks inthe molecule. Typically, (u) is greater than 50, preferably between 50and 150, more preferably between 80 and 130. The PPO blocks are of thegeneral formula;

—(—C₃H₆—O—)_(v)—  (3)

[0055] where (v) may be the same or different for different PPO blocksin the molecule. Typically, (v) is greater than 25, preferably between25 and 75, and more preferably is between 30 and 60.

[0056] The block copolymers may be branched structures and include otherstructures (e.g. bridging structures, or branching structures) andsubstituents that do not materially affect the ability of the blockcopolymer to adsorb upon and cover a hydrophobic surface.

[0057] In one embodiment, the block copolymer surfactant used to makeEGAP is a polymeric tri-block copolymer with pendant —OH groups, as inFormula (4) below. These tri-block copolymers have a hydrophobic centerblock of polypropylene oxide and hydrophilic end blocks of polyethyleneoxide with terminal —OH groups, and can be represented by the formula:

HO—(—C₂H₄—O—)_(x)—(—C₃H₆—O—)_(y)—(—C₂H₄—O—)_(z)—H  (4)

[0058] where (y) is between 25 and 75, preferably between 30 and 60, and(x) and (z) are preferably the same but may be different, and arebetween 50 and 150, preferably between 80 and 130. Block copolymersurfactants of the type described are commercially available from, forexample, BASF.

6.2.2 Activation of the End-Group of a Polymer to Yield an EGAP

[0059] The end-group of the polymer is activated by methods well knownin the art. Briefly, the —OH end groups of the PEO chains of thepolymeric surfactant are modified to introduce a small reactive organicgroup which is stable in water. Using the block copolymer surfactantsrepresented by equation (4) as an example, if both —OH groups on thependant PEO chains are substituted, the modified surfactant has theformula;

R—O—(—C₂H₄—O—)_(x)—(—C₃H₆—O—)_(y)—(—C₂H₄—O—)_(z)—R  (7)

[0060] where R is a reactive group. Accordingly, the general formula forthe modified polymeric surfactants of the invention is:

(HO—PEO)_(c)(R—O—PEO)_(d)(PPO)_(b)  (8)

[0061] where (c+d) is equal to (a) in formula (1), and (c) is 0 or apositive integer, and (b) is defined above for formula (1). The R groupmay be any reactive group that is stable in water and will impart thedesired selective reactivity for the substrate surface when the modifiedsurfactant is adsorbed upon the surface. The specific reactivity may beto any non-water entity or entities.

[0062] The R groups are chosen such that they do not significantlyimpair adsorption of the modified polymeric surfactant on thehydrophobic surface. For example, in a preferred embodiment of theinvention, the reactive R group contains a hydrazino group (by furtherreacting a p-nitrophenyl group), a thiopyridyl group, a tyrosyl residue,or a maleimide. R may also be a member of the group consisting ofhydrozino, thiopyridyl, tyrosyl, malcimide, 2-pyridyl disulphide,5-nitro-2-pyridyl disulphide, 4-pyridyl disulphide, 5-carboxy-2-pyidyldisulphide, and the nitrogen oxides of 2-pyridyl disulfide,5-nitro-2-pyridyl disulfide, 4-pyridyl disulfide, and 5-carboxy-2-pyidyldisulphide as well as other groups well known in the art.

[0063] In another embodiment the R group is for the immobilization ofbiomolecules and contains the structure:

—S—S—R″  (9)

[0064] where R″ is selected from the group consisting of (1)2-benzothiazolyl, (2) 5-nitro-2-pyridyl, (3) 2-pyridyl, (4) 4-pyridyl,(5) 5-carboxy-2-pyridyl, and (6) the N-oxides of any of (2) to (5). SeeU.S. Pat. Nos. 4,149,003 to Carlson et al. and 4,711,951 to Axen et al.which are hereby incorporated by reference.

6.3 Binding EGAPs to Hydrophobic Surfaces

[0065] Once the EGAP is formed, the EGAP is adsorbed onto an appropriatehydrophobic surface. This simply requires mixing the appropriate amountof EGAP with the hydrophobic surface. Usually approximately two hours issufficient to completely coat the hydrophobic surface with EGAP.Depending on the shape and size of the hydrophobic surface to be coated,it may be advantageous to shake the mixture to ensure that the entiresurface area becomes coated.

[0066] It will be appreciated that the concentration of EGAP can beregulated by diluting the EGAP with polymer that has not been activated(i.e., block copolymer surfactants). In this way, the number of EGAPreactive sites, and hence, conjugated biomolecules can be regulated. Inaddition, by using a block copolymer surfactant to dilute the EGAP, thesurface does not adsorb cell, proteins and other biomolecules. Forexample, as illustrated in FIG. 2, various hydrophobic material surfacescoated with a unmodified block copolymer surfactants such as Pluronic™F108 substantially decrease the adhesiveness of the surface to NIH 3T3cells.

6.4 Suitable Biomolecule Conjugates

[0067] It will be appreciated by one skilled in the art that there is alarge number of biomolecules that can be conjugated to EGAP according tothe composition and the method of the present invention. As used hereinthe term biomolecule refers to any molecule that can be conjugated toEGAP, including, but not limited to, proteins, peptides amino acids,nucleic acids, lipids, carbohydrates, and combinations thereof. Thebiomolecules can be native, recombinant, or synthesized. In fact, theterm biomolecule as it is used herein is not limited to naturallyoccurring molecules, but includes molecules such as syntheticpharmaceutical drugs which have no biological origin.

[0068] In a preferred embodiment, the biomolecules are ECM proteins,adhesion proteins, growth factors, or other biomolecules generally usedin cell culture. Below is an exemplary review of some of thebiomolecules that can be conjugated to EGAP and used according to thepresent invention.

6.4.1 Extracellular Matrix Proteins

[0069] EGAP could be conjugated to an ECM protein. For example, EGAPcould be conjugated to one or more of the various collagen moleculescurrently known or hereafter isolated. Collagen is the name given to asuperfamily of ECM proteins whose primary role is forming and preservingthe structural integrity of the ECM and cells. Collagen's characteristictriple-helix domain forms fibrils, filaments, or networks, either aloneor in combination with other ECM components.

[0070] Collagen IV is a major component of basement membranes and formsa network to which other basement components, such as laminin, nidogen,heparin and heparan sulfate proteoglycans, interact. Many cells typesadhere to Type IV collagen. See, e.g., Glansville, R. W. “Structure andFunction of Collagen Types,” Academic Press Inc., pp. 43-79. Moreover,regions within Type IV collagen are known to promote or inhibit celladhesion. Vandenberg et al., J. Cell Biol., 113: 1475-1483 (1991);Tsilibary et al., J. Cell Biol. 111: 1583-1591. Surfaces coated withType IV collagen (or specific regions or Type IV collagen) conjugatedEGAPs, therefore, could be used to both promote and inhibit cellattachment and growth in vitro.

[0071] Type IV collagen can be obtained from basement membranes treatedwith pepsin or with bacterial collagenase. Glansville, R. W. “Structureand Function of Collagen Types,” Academic Press Inc., pp. 43-79; Hudson,et al., “Extracellular Matrix Macromolecules—A Practical Approach” (M.A. Haralson and J. R. Hassell, eds.) IRL Press, in press. Moreover, thecomplete primary structures of mouse and human a(IV) and a2(IV) chainshave been deduced from cDNA sequences and mouse and human a(IV) anda2(IV) genomic clones have been extensively characterized. Vuorio etal., Annu. Rev. Biochem. 59: 837-872 (1990); Sandell, L. J. and Boyd, C.D., “Extracellular Matrix Genes” (L. J. Sandell and C. D. Boyd, eds.)Academic Press Inc. pp. 1-56 (1990); Blumberg, B. and Kurkinen, M.“Extracellular Matrix Genes (L. J. Sandell and C. D. Boyd, eds.)Academic Press Inc., pp. 115-135 (1990). The corresponding polypeptidescoded by these genes, therefore, could be obtained using recombinanttechniques well known in the art.

[0072] In another example, the biomolecule could be a fibrillar collagenwhich includes five different molecular types (I, II, III, V, and XI).Fibrillar collagens polymerize to form fibrils that serve as stabilizingscaffolds in extracellular matrices. Cell attachment, differentiation,and migration are influenced by fibrillar collagens. It has been shownthat fibrillar collagens interact with cells through receptors on thecell surface. See e.g., Hemler, M. E., Annu. Rev. Immunol. 8: 365-400(1990).

[0073] Surfaces coated with fibrillar collagen conjugated EGAPs,therefore, would promote cell attachment, differentiation and migrationand better mimic in vivo biological conditions. Fibrillar collagen iscommercially available from, for example, Sigma, St. Louis, Mo.Moreover, methods of purifying, as well as cDNAs coding fibrillarcollagen, are well known in the art. (See e.g., Vuorio, et al. Annu.Rev. Blochem. 59: 837-872 (1990).)

[0074] EGAP could also be conjugated to one or more fibronectinmolecules or peptides thereof. The subunits of fibronectins vary in sizebetween approximately 235 and 270 kDa plus carbohydrates. Extendedpolypeptide segments in certain parts of the molecule are highlysusceptible to proteolysis, which generates a series of proteaseresistant domains, each comprising several of the repeating modules.These domains contain a variety of binding sites for other molecules,including collagens, fibrin, heparin/heparan sulphate, and cell surfacereceptor integrins.

[0075] Fibronectins are widely expressed in embryos and mature cells,especially in regions of active morphogenesis, cell migration, andinflammation. Fibronectins promote the adhesion and spreading of manycell types by binding to several different integrin receptors. See,e.g., Hynes, R. O., Cell 48:549-554 (1987). Tumor cells show reducedlevels of fibronectin and levels in plasma fall in various forms oftrauma. In contrast, fibronectin levels are elevated during woundhealing and fibrosis.

[0076] Fibronectin conjugated EGAPs, therefore, could be used in tissueculture to assist in cell adhesion, morphogenesis, and cell migration.Moreover, biomaterials, such as surgical wraps, could be coated withfibronectin conjugated particles to aid and accelerate wound healing.

[0077] The full length polypeptide of fibronectin, like many otherproteins, is not required for many of the activities and propertiesdescribed above. For example, it is known that fibronectin has two cellbinding sites which are recognized by two different integrin receptors.The first cell binding site comprises three residues:arginine-glycine-aspartic acid, or RGD. The second cell binding sitecomprises the peptide: glutamic acid-isoleucine-leucine-asparticasid-valine, or EILDV. Many of these peptide, including RGD, RGDS, RGES,RFDS, GRDGS, and GRGS are commercially available from, for example,Sigma, St. Louis, Mo.

[0078] Other peptides such as GRGDTP inhibit cell attachment offibronectin, vitronectin, and Type I collagen. Amino acid sequenceQPPRARI is the binding site for the carboxy-terminal heparin bindingdomain. Peptides that inhibit platelet aggregation and inhibitfibronectin binding to bacteria are also well known and commerciallyavailable. EGAP, therefore, can be simply conjugated with any number ofpeptides or domains to obtain the desired results according to thepresent invention.

[0079] In another example, EGAP could be conjugated to agrin. Agrin isECM glycoprotein which can take the form of either a 150 kDa or a 95 kDaprotein. Agrin is localized at the neuromuscular junction and inducesclustering of acetylcholine receptors on skeletal myotubes in cellculture. Clustering of this receptor is one of the most dramatic eventsin neuromuscular synapse formation and regeneration in vivo. Purifiedagrin has been shown to induce clustering of synaptic molecules invitro, such as ECM-associated acetylcholinesterase andmembrane-associated acetylcholine receptors, and it is very likely tofunction similarly in vivo.

[0080] As an agent that induces differentiation in skeletal myotubes,agrin is synthesized in motor neurons and transported to their terminalsin skeletal muscles. Agrin has been shown to induce phosphorylation ontyrosine on the acetylcholine receptor β-subunit. Treatments thatinhibit receptor aggregation prevent tyrosine phosphorylation. Resultssuggest that the agrin receptor regulates a tyrosine protein kinase orphosphatase that in turn regulates receptor clustering. These and otherdata demonstrate that the extracellular matrix protein agrin containsall the essential information needed to form a neuromuscular synapse.Therefore, the ability to conjugate agrin to EGAP to form an agrinconjugated EGA would be a significant advancement in co-culture celltechnology. This, together with the fact that conjugated block copolymersurfactant surfaces do not adsorb secreted cellular products that couldbe important for differentiation and growth, would approximate an invitro system to study the formation and function of the neuromuscularjunction. Understanding the formation of the neuromuscular junction isthe first step in understanding the formation of more complex synapsesin the central nervous system.

[0081] Agrin can be obtained from the basal-lamina enriched fraction ofT. Californica electric organ as described in Nitkin et al., J. CellBiol., 105: 2471-2478 (1987). Alternatively, portions of agringlycoprotein can be obtained from available cDNAs using recombinanttechniques well known in the art. Tsim et al., Neuron, 8: 677-689(1992). Monoclonal antibodies against T. Californica agrin are alsoavailable. Reist et al. J. Cell. Biol., 105: 2457-2469 (1987).

[0082] In addition, one skilled in the art will appreciate that otherECM components and proteins, such as aggrecan, biglycan, bonesialoprotein, cartilage matrix protein, Cat-301 proteoglycan, CD44,cholinesterases, FACIT collagens (Type IX, XII, XIV), other collagens(Type VI, VII, XIII), short chain collagens (Type VIII, X), decorin,elastin, fibrinogen, fibroglycan, fibromodulin, fibulin, glypican,HB-GAM, hyaluronan and hyaluronan binding proteins, J1 glycoproteins,laminin, laminin binding proteins, link protein, mucins,nidogen/entactin, osteopontin, perlecan, plasminogen, plasminogenactivator inhibitor 1, plasminogen activator inhibitor 2, proteinscontaining Ca²⁺-dependent carbohydrate recognition domains, restrictin,serglycin, SPARC/osteonectin, syndecan, tenascin, thrombospodin,tissue-type plasmogen activator, urokinase type plasminogen activator,versican, vitronectin, and von Willebrand Factor could be conjugated toEGAP and used in numerous biological processes and assays.

6.4.2 Cell Adhesion Molecules

[0083] Cell adhesion molecules are molecules that, in addition tomediating cell-ECM contact, mediate cell-cell contact. Cell adhesion isrequired at all stages in development and critical to the overallorganization of tissues and organs. To date, there have been hundreds ofcell adhesion molecules isolated and characterized. These molecules canbe roughly categorized into five protein superfamilies: (1) theimmunoglobulin (Ig) superfamily; (2) the cadherin superfamily; (3) theintegrin superfamily; (4) the selectin superfamily; and (5) the H-CAMsuperfamily.

[0084] The Ig superfamily, for example, which includes other proteinssuch as CAM and N-CAM are primarily involved in cellular recognition. Assuch, immunoglobulins or antibodies play a critical role in properimmune function. See Williams et al., Ann. Rev. Immunol. 6: 381-405(1988). As will be discussed below, the Ig superfamily of moleculesforms the bases for immunoassays in the art.

[0085] The feasibility and value of conjugating adhesion protein of theimmunoglobulin superfamily to EGAP has been demonstrated. U.S. Pat.No.5,516,703. In one embodiment, a significant increase in antigenbinding was observed when anti-IgE was immobilized to a polystyrenesurface via an EGAP tether rather than immobilization through simpleadsorption as is the common practice in the art today. In fact, antigenbinding to anti-IgE conjugated EGAP was on average 4 times greater thatanti-IgE adsorbed to the PS surface.

[0086] The same strategy could be used to conjugate numerous adhesionproteins to EGAP for the purposes of biological analysis or cell growth.Examples include AMOG, cadherins, CD2, CD4, CD8, C-CAM (CELL-CAM 105),cell surface galactosyltransferase, connexins, desmocollins, desmoglein,fasciclin I, fasciclin II, fasciclin III, F11, GP Ib-IX complex,integrins, intercellular adhesion molecules, L1, leukocyte commonantigen protein tyrosine phosphate (LCA, CD45), LFA-1, LFA-3, mannosebinding proteins (MBP), MUC18, myelin associated glycoprotein (MAG),neural cell adhesion molecule (NCAM), neurofascin, neruoglian,neurotactin, PECAM-1, PH-20, selectins, TAG-1, VCAM-1 and the like. Infact, hundreds of immunoglobulin proteins directed at different cellularcomponents are commercially available from, for example, Sigma ChemicalCOW'S., St. Louis, Mo.

6.4.3 Growth Factors, Mitogens, and Differentiation Factors

[0087] Cell contact with a number of different biomolecules, such asgrowth factors, mitogens, and differentiation factors can stimulate celldivision. Many of these biomolecules are normally present in growthserum used in most cell culture media. Others biomolecules, like somebacterial lipopolysaccharides and certain cell agglutinating proteins(lectins), are not present in normal growth media. Nevertheless, theyare an integral part of work aimed at discerning what signals regulatecell growth and thus have huge implications in cancer research.

[0088] For example, platelet-derived growth factor (PDGF), would be anideal candidate to conjugate to EGAP. PDGF is the major polypeptidemitogen in cell culture serum. In fact, studies in fibroblastdemonstrate that PDGF is required to make cells competent to othergrowth factors. That is, the cell will not respond to other growthfactors and mitogens unless they are first exposed to PDGF.

[0089] In vivo, PDGF is stored in the α granules of the blood platelets.During blood clotting and platelet adhesion, PDGF is released from theseα granules. The release of PDGF at the site of injury causes some celltypes to migrate to the site of injury and causes other cell types todivide. Accordingly, PDGF conjugated EGAPs could be used to promote cellgrowth in culture. In addition, PDGF could be used to promote woundhealing in vivo by applying it to wounds in conjunction with anappropriate biomaterial. The PDGF would be tethered to EGAP which inturn would be adsorbed to the biomaterial. Thus, unlike other drugdelivery methods, the amount of PDGF stimulation and the site ofstimulation could be tightly regulated.

[0090] In addition, one skilled in the art will appreciate that othergrowth factors and mitogens, such as EGF, TGF-α, TGF-β, NGF, IGF-I,IGF-II, GH, and GHRF can also be conjugated to EGAP according to themethod of the present invention and be used for cell culture and avariety of biological assays.

[0091] Another group of biomolecules that could be conjugated to EGAPare differentiating factors. These factors determine the fate ofprecursor cells, such as stem cells. For example, depending on thedifferentiating factor or factors which a stem cell is exposed, a stemcell can become a plasma cell, a memory B lymphocyte, an activated Tcell, a macrophage, blood platelets, or a erythrocyte.

[0092] These factors, therefore, have tremendous implications in vitroand in vivo. In vitro, stem cells could be grown on one or more of thesefactors conjugated to EGAP. As such, the fate of the cell can becarefully controlled. For example, the ability to regulate T cellproduction in vitro from precursor cells could be used to supplement theloss of T cells that leads to acquired immunodeficiency syndrome (AIDS).In vivo, the differentiating factor erythropoietin is currently beingused to increase red blood cell production in patients that have lostlarge volumes of blood.

[0093] In addition, one skilled in the art will appreciate that otherdifferentiating factors and proteins, such as multi-CSF (II-3), GM-CSF,G-CSF, and M-CSF can also be conjugated to EGAP.

6.4.4 Nucleic Acid

[0094] Nucleic acids can also be conjugated to EGAPs. Nucleic acids asthe term is used herein refers to molecules comprised of natural andsynthetic DNA and RNA molecules. One skilled in the art will appreciatethat DNA and RNA can be modified or conjugated without disturbing itsbiological activity. Moreover, nucleic acids of various lengths can beeasily synthesized and linked together using synthesis and ligationtechniques commercially available and well known in the art.

[0095] A common strategy in the art is to substitute one of thenucleotides or bases in a DNA with a universal base. A recentpublication, for example, describes the properties of 3-nitropyrrole2′-deoxynucleoside when used as universal nucleoside. Briefly,3-nitropyrrole 2′-deoxynucleoside can be used for many purposes,including sequencing, PCR, ligase chain reaction, in situ hybridization,mutagenesis, motif cloning, and even in RFLP. 3-Nitropyrrole2′-deoxynucleoside is commercially available form, for example,Bio-Synthesis, Lewisville, Tex.

[0096] In a preferred embodiment, the nucleic acid is modified with afree thiol group. The free thiol group has been shown to be reactivetowards maleiimide or an iodoacetyl-derived conjugate. Binding ofalkaline phosphatase, horseradish peroxidase, and various fluorophoresto synthetic oligonucleotides by means of a free thiol group has beenreported in the literature. Nucleic acids with 5′ thiol C6, 3′ thiol C3S—S, and 5′/3′ thiol C6 S—S base modifications are commerciallyavailable form, for example, Bio-Synthesis, Lewisville, Tex.

[0097] Also, commercially available are technologies for attachingreactive amine groups at the 5′ terminus, 3′ terminus, or any internalposition. The Amino-I, and Amino-II can incorporate a primary aliphaticamine functional groups into oligonucleotides at single or multiplesites. Many of these analog are suitable for attaching the DNA to othermolecules, e.g., EDTA or alkylating reagents which can cut thecomplementary strand or double strand. Other examples include 5′-C3amine, 5′-C12 amine, 3′-C3 amine, 3′-C7 amine, amino C6 dT, amino I,amino II, 3′-DMT-C6 amine, amino C2 dT which are commercially availablefrom, for example, Bio-Synthesis, Lewisville, Tex.

[0098] Vaious other base modifications include deoxy inosine (dI), deoxyuridine (dU), 5-methyl-dC, O-6-ME-dG, 5-I-dU,5-I-dC, 5-Br-dU,3-nitropyrrole (M), 3′-dA (cordycepin), 2′, 3′-ddC, TMP-F-dU,04-triazolyl-dT, 06-phenyl-dI, 2-aminopurine, 04-triazolyl-dU, 7-deazadG, N-6-Me-2′dA, S6-DNP-dG, 5′-OMe-dT, ethano-dA, 5′ or 3′phosphorylation, 3′-spacer C3, carboxy-dT are commercially availablefrom, for example, Midland, Midland, Tex. and Bio-Synthesis, Lewisville,Tex.

6.5 Biomolecule Conjugated EGAP

[0099] Biomolecules can be conjugated to EGAP using numerous methodsknown in the art. By reacting hydroxylated block copolymer surfactantswith 4-nitrophenyl chloroformate, one can efficiently conjugatebiomolecules having a variety of reactive groups. For example, EGAPsreact relatively easily in an organic solvent with amino groups,2-pyridyl disulfides, peptide, hydrazino and other amino containingmolecules. Using hydrazino groups as the bridge, tyrosyl groups forradioisotope labeling purpose can be subsequently coupled to the EGAP bya reaction with the Bolton-Hunter reagent.

[0100] Biomolecules are conjugated via amine groups. In one embodiment,4-nitrophenyl chloroformate activated EGAP was conjugated to abiomolecule via an amine group on a peptide. The peptideglycyltryptophan (Gly-Trp) was mixed with an appropriate amount of4-nitrophenyl chloroformate activated EGAP. The two compounds wereallowed to react at 25° C. overnight. The reaction mixture was thenpurified by passing it through a Sephadex column. Gly-Trp conjugatedEGAP was confirmed by dry weight and photometric analysis.

[0101] Biomolecules can also be conjugated to EGAP via a disulfide bond.EGAP molecules, as discussed above, can be activated by introducing areactive group containing a disulfide derivative such as a2-(2-pyridyldithio)ethylamine. This method of conjugation is preferredbecause the rate of hydrolysis of the 2-pyridyl disulfides groups atabout pH 8.5 is almost negligible in comparison to the rate of thethiol-disulfide exchange reaction. As such, only a small concentrationof biomolecule is required.

[0102] Moreover, this conjugation method provides an easy way to detectthe degree of biomolecule conjugation. The reaction between the thiolgroup on the biomolecule and 2-(2-pyridyldithio)ethylamine releasesthiopyridone. Thiopyridone concentration can be readily and accuratelyquantified by spectroscopic detection at 343 nm with an extinctioncoefficient of 8060/cm⁻¹M⁻¹. Thus, the concentration of thiopyridone isdirectly proportional to the degree of biomolecule conjugation.

[0103] Finally, since the thiol-disulfide exchange is a reversiblereaction, bound biomolecules can be released from the solid phase byaddition of a thiol-containing reagents, such as dithiothreitol (DTT).

[0104] In one embodiment, the biomolecule fibronectin and human serumalbumin was conjugated to EGAP using the methods described above. Asillustrated in FIGS. 4 and 5, microcalorimetry studies indicate thatthese biomolecules retain their native secondary structure when tetheredto EGAPs.

6.6 Uses for Biomolecule Conjugated EGAP Coated Surfaces

[0105] It will be appreciated by one skilled in the art that given thelarge number of biomolecules that can be conjugated to EGAP, the numberof uses for biomolecule conjugated EGAPs is also large. Below areexemplary uses for biomolecule conjugated EGAPs.

6.6.1 Method of Attaching and Growing Cells

[0106] The composition and the method of the present invention can beused to attach and grow cells in culture. As discussed above, EGAP canbe conjugated to any number of ECM and cell adhesion proteins as well asgrowth and differentiation factors.

[0107] In one embodiment, NIH 3T3 cells were grown on hydrophobicculture surface coated with GRGDSY conjugated EGAP. GRGDSY is a peptidecorresponding to a cell binding site of fibronectin. The GRGDSY wasconjugated to an F108 derivative EGAP via a disulfide bond preparedaccording to the 2-pyridyl disulfide conjugation method described above.Once the GRGDSY was conjugated to EGAP and the hydrophobic surface waswashed, NIH 3T3 cells were seeded at a concentration of 6×10³ cells/cm²in DMEM supplemented with 10% bovine serum. Qualitatively, as illustratein FIG. 5, fibroblast cells were in good health and were able to attachextend processes in GRGDSY conjugated EGAP. On the contrary, asillustrated in FIG. 6, no attachment was observed on cells seeded onunconjugated F-108.

[0108] Quantitatively, as illustrate in FIG. 7, nearly the same numberof cells attached to GRGDSY conjugated EGAP (PDSF108/RGD) as attached tountreated polystyrene (PS). Moreover, cell attachment and growth isdirectly related to the GRGDSY conjugated EGAP as very littleattachment, if any, was found when the surface was coated with F-108(F108), F-108 containing unconjugated GRGDSY (F108/RGD), and 2-pyridyldisulfide conjugated F-108 (PDSF108).

[0109] It will be appreciated by one skilled in the art that the sameprinciples and methodologies could be used to grow other cells,including other eukaryotic cells such as insect cells, yeast and plantcells, and prokaryotic cells such as bacteria.

6.6.2 Method for Selecting and Sorting Cell and Other BiologicalMaterial

[0110] The composition and the method of the present invention can beused to sort cells and other biological material. It will be appreciatedby one skilled in the art that it is often desirable to select one celltype from a mixture of cells. For example, identifying lymphocytes aseither T cells or B cells is useful in diagnosing various diseases,including lymphoproliferative malignancies, immunodeficiency diseases,unexpected infections diseases, monitoring of transplants, and acquiredimmunologic disorders such as AIDS. Current methods involve acombination of density gradient centrifugation and either fluorencencemicroscopy or cell flow cytometry (or fluorescence-activated cellsorter). These methods are tedious and expensive. Moreover, the stressof the procedure often damages the cells making it difficult, if notimpossible, to grow the cells once they have been selected.

[0111] The method of the present invention could be used to quickly sortcells. EGAP could be conjugated to a number of biomolecules that arespecific for the desired cells type. For example, the biomolecule couldbe a monoclonal antibody against a specific cell surface antigen such asa transmembrane receptor or a particular carbohydrate moiety. Many ofthese biomolecules, including CD2, CD3, CD4, CD8 on T cells and CD 19,CD20, CD2, and surface immunoglobulins on B cells are all commerciallyavailable from, for example, Sigma Chemical Company, St. Louis, Mo.

[0112] In one embodiment, the cell specific biomolecule conjugated EGAPis coated on polystyrene beads. The polystyrene beads are then combinedwith a mixture of cells under appropriate incubation conditions andgrowth media that does not contain molecules that will bind to the cellspecific biomolecule. After the cells have had an opportunity to bind tothe cell specific biomolecule, the polystyrene beads are separated fromthe remaining unattached cells. The separation means is any means wellknown in the art including magnetic, streptavidin separation, ormechanical separation such as gentle centrifugation. In one embodiment,a subpopulation of the EGAPs coated to the polystyrene beads comprisesbiotin conjugated EGAP. Therefore, the biotin is available for bindingand separation with streptavidin, such as streptavidin MagneSpheres®paramagnetic particles sold by Promega, Madison, Wis. After severalgentle washes to remove non-specifically bond cells, the collectedpolystyrene beads are assayed directly using common bioassays well knownin the art or cultured. These methods of cell sorting are only exemplaryof the many cell sorting methods that can be used with the compositionand method of the present invention.

6.6.3 Biological Assays 6.6.3.1 Immunoassays

[0113] As discussed above, after infection with a pathogen, the immunesystem recognizes the pathogen as foreign and begins to produce largequantities of antibodies against the pathogen. The antibodies bind tothe pathogen and initiate other immune functions which are aimed ateliminating the pathogen from the organism.

[0114] Thus, a common way of determining whether a given individual isinfected with a certain pathogen, such as HIV, is to assay for thepresence of antibodies against HIV in the individual's blood. There aremany types of immunoassays known in the art. The most common types ofimmunoassay are competitive and incompetitive heterogeneous assays suchas enzyme-linked immunosorbent assays (ELISA). In immunoassays thereactant is an antigen. In a noncompetitive ELISA, unlabeled antigen iscommonly bound to a hydrophobic surface through adsorption. Biologicalsample is combined with antigens bound to the surface and antibodies(primary antibodies) in the biological sample are allowed to bind to theantigens forming immune complexes. After immune complexes have formed,excess biological sample is removed and the reaction cells are washed toremove nonspecifically bound antibodies. Immune complexes are thenreacted with an appropriate enzyme-labeled anti-immunoglobulin(secondary antibody). Anti-immunoglobulins recognize bound antibodies,but not antigens. Anti-immunoglobulins specific for antibodies ofdifferent species, including human, are well known in the art andcommercially available from Sigma Chemical Company, St. Louis, Mo. andSanta Cruz Biotechnology, Santa Cruz, Calif. After a second wash step,the enzyme substrate is added. The enzyme linked to the secondaryantibody catalyses a reaction which converts substrate into product.When excess antigen is present, the amount of catalyzed product isdirectly proportional to the amount of antigen specific antibodies(analyte) in the biological sample. Typically, the reaction product iscolored and thus measured spectrophotometrically using UV/VIS technologyand equipment well known in the art.

[0115] Biomolecule conjugated EGAP's are suitable for immunoassaytechnology as illustrated by data using IgE.

6.6.3.2 Method of Immobilizing Virus for Analysis

[0116] The present invention may also be used to collect viruses forvarious uses, including growth and bioassay. For example, as discussedabove, a common way of detecting whether an individual has been infectedwith a particular pathogen was to assay for antibodies against thepathogen in the individual's blood. Many times, however, this techniqueis unsuitable. For example, diagnosing of HIV infection in infants isdifficult due to the placental passage of IgG antibodies from theinfected mother to the child. Moreover, there is a window between thetime an individual becomes infected with a pathogen and the developmentof a detectable antibody producing immune response. Using immunoassays,an individual who is in fact HIV positive may test negative for HIVbecause the level of HIV specific antibodies are not detectable when thetest is administered. Therefore, infants and some other individuals aretested for HIV infection using PCR techniques, a sensitive techniquewhich assay for HIV DNA rather than antibodies against HIV.

[0117] In order to assay for HIV DNA, a quantity of virus must beobtained. Currently this is done by taking a small portion of theindividual's blood. However, the amount of non-viral DNA in theindividual's blood decreases the sensitivity, specificity, and ibackground of the assay. It would be an advantage, therefore, to enrichthe sample for virus before a DNA-based assay such as PCR is performed.

[0118] The present invention provides such means. EGAP adsorbed to ahydrophobic surface is conjugated to a biomolecule which is specific forHIV such as antibodies against gp 120, a glycoprotein which is expressedon the surface of the virus. The individual's blood is, for example,passed through a column containing polystyrene beads adsorbed with gp120conjugated EGAP. HIV viruses bind to gp 120 conjugated EGAP while otherblood components pass through the column. After a series of low saltwashes, the polystyrene beads containing bound HIV are assayed using PCRtechnology well known in the art.

[0119] Generally, oligonucleotide primers to conserved regions of HIVgenes, such as the gag and pol genes, are synthesized and used toamplify a region of the viral gene. The amplified PCR product is thendenatured and a radiolabelled DNA probe is added and permitted tohybridize with the amplified product. The hybridized product isidentified by running the mixture on a polyacrylamide gel followed byautoradiography.

[0120] Recently, PCR techniques employing tris-bipyridineare ruthenium(II) complexes have greatly facilitated the procedure and sensitivity ofPCR techniques and thus are also contemplated by the present invention.See Kenten, J. H., et al. “Rapid Electrochemiluminescence Assays ofPolymerase Chain Reaction Products”, Clin. Chem., 37: 1626-1632 (1991);T. E. Schutzbank & J. Smith, “Detection of Human Immunodeficiency VirusType 1 Proviral DNA by PCR Using an Electrochemiluminescence-taggedProbe,” J Clin Microbiol 33: 2036-2041 (1995) which are herebyincorporated by reference. Briefly, oligonucleotide primers directed atconserved regions of an HIV gene are synthesized and used to amplify aregion of that gene. One of the oligonucleotides is biotinylated (linkedto a biotin molecule) by methods well known in the art. The amplifiedPCR product is then denatured and hybridized with an ECL-labeled DNAprobe which is complementary to the amplified biotinylated DNA stand.After an appropriate hybridization period, thebiotinylated-DNA/ECL-labeled DNA hybrid is reacted with streptavidincoated magnetic particles. A magnetic force is applied to retain thebiotinylated-DNA/ECL-labeled DNA hybrids in the reaction vessel whileunhybridized material is removed. Finally, the ECL complexes are excitedby chemical, photometric, or electrical means and the photon emissionmeasured.

[0121] The above example is merely exemplary for how the presentinvention can be used to enrich biological samples for viruses for usein biological assays and for growth using techniques well known in theart.

7. EXAMPLES

[0122] The following examples are given to illustrate variousembodiments which have been made with the present invention. It is to beunderstood that the following examples are not comprehensive orexhaustive of the many types of embodiments which can be prepared inaccordance with the present invention.

Example 1 Coupling of Amines to EGAP

[0123] (A) 1,3 Diaminopropane. 1,3 Diaminopropane (3.3 g) was mixed with5 mL of deionized water. After the pH was adjusted to 8.2 withconcentrated HCL, the solution was mixed with a solution of 0.5 g of4-nitrophenyl chloroformate activated Pluronic™ F108 in 5.0 mL ofdeionized water. The reaction mixture, which immediately turned yellow,was kept at 25 C for 15 h. This solution was transferred to a dialysistubing (with a molecular weight cutoff of 3500) and was dialyzed against4 L of deionized water. During the 48 h dialysis process, water waschanged five times until the low molecular weight material was assumedto be completely removed. The product was then recovered bylyophilization. The degree of substitution was determined by elementalnitrogen analysis. In this calculation the nitrogen content determinedper a given mass of product was taken to exclusively derive from theattached diamine.

[0124] (B) 2-Aminoethanesulfonic Acid (Taurine). Taurine (3.4 g) wasdissolved in 7 mL of deionized water, and the pH solution was adjustedto 9.4 with 2M Hcl. The solution was mixed with a 5 mL water solution of0.5 g of 4-nitrophenyl chloroformate activated Pluronic™ F108. Theresulting reaction mixture was kept at 25 C overnight, and the productwas obtained after dialysis and lyophilization as described previously.The degree of substitution was determined through sulfur and nitrogenanalysis of a known amount of product; its molar taurine content wasthen readily calculated.

[0125] (C) Glycyltryptophan (Gly-Trp). Two 11 mg portions of Gly-Trpwere each dissolved in a vial with 2 mL of methanol. One of the vialscontained 0.05 mL of 1.2 M TEA. To both vials was added 11 mg of4-nitrophenyl chloroformate activated Pluronic™ F108, and the finalsolutions were kept at 25 C overnight. The reaction mixtures were thenpassed through PD-10 Sephadex G-25 columns, and the void fractions werepooled. The amount of bound Gly-Trp was determined by dry weightdetermination and photometric analysis using a molar extinctioncoefficient of 6170 cm⁻¹ M⁻¹ for the tryptophan residue.

Example 2 Calorimetric Observations of Fibronectin Conjugated to EGAP

[0126] PS latex particles with a diameter of 261 nm were purchased as a10% (w/v) suspension were purchased from Seradyn, [State]. The blockcopolymer surfactant surfactant used was F108 having a molecular weightof 14600 were donated by BASF COW'S., [state].Nsuccinimidyl-3-(2-pyridyldithiol) propionate (SPDP) was obtained fromPierce, [state]. Diihiothreitol (DTT) was from Bio-Rad, [state].Fibronectin solution (FN, 1.5 mg/ml) was isolated from human plasma, anddisposable prepacked PD-10 columns were purchased from Pharmacia, Wis.

[0127] The Pluronic™ F108-2-pyridyl disulfide derivative (GAP) wassynthesized as described above.

[0128] Adsorption reaction was carried out in a mixture consisting of 10μL of the suspension of PS latex particles and 200 μL of 0.5% (w/w) EGAPdissolved in deionized water. This mixture was incubated for 2 hourswith shaking at room PS microspheres were washed and recovered by tablecentrifugation Eppendorf 5415C).

[0129] Thiolation of biomolecules the methodology described previously(4). Briefly, the reaction mixture of 2 mL of fibronectin solution and20 pL of 5 mM SPDP solution was kept for 1 hour at room temperature withshaking, after which it was passed through a PD-10 column. IleSPDP-modified fibronectin (FN-SPDP) was collected; its emergence fromthe PD-10 column was monitored by UV adsorbance at 280 nm. Thiolatedfibronectin was then obtained by adding 4 liL of a 50 mM D7r solution tothe SPDP-modified fibronectin and keeping the mixture for 30 minutes atroom temperature. The sulfbydryl concentration was calculated byquantifying the concentration of released 2-thiopyridone as describedearlier.

[0130] Low molecular weight reaction products were removed by passingthe thiolated fibronectin (FN-SH) reaction mix through a PD-10 column.The coated PS latex particles were added to FN-SH or FN solution and thelinking reaction was allowed to take place for 1 hour at roomtemperature under shaking. The latex particles were washed andcharacterized by means of differential scanning microcalorimetry (DSC).Quantification of the amount of FN or FN-SH bound onto the latexparticles was performed by amino acid analysis as well as by Micro BCAassay, as described before. DSC (Hart Scientific, Model 4207) studieswere carried out as reported previously .

[0131] FN shows melting transitions at different temperatures and withdifferent enthalpic contents. It is therefore likely that surfaceadsorption might strongly affect the FN structure. FIG. 3 is acomparison of FN adsorbed and bound through the PEO tether offered bythe modified Pluronic™ F108, respectively. No cooperative transitionsare in evidence for the adsorbed protein, while FN tethered to thesurface shows the normal, complex transition pattern. Apparenttransition enthalpies for free and immobilized FN are listed in Table 1.TABLE 1 Fibronectin T_(m) (° C.) ΔH(K cal/mol) In PBS 55-80 570Conjugated to 55-80 440 EGAP

Example 3 Calorimetric Observations of Human Serum Albumin Conjugated toEGAP

[0132] Human Serum Albumin (HSA) conjugated EGAP was thiolated,purified, and conjugated to EGAP essentially as described in Example 2.The attachment of HSA to a PEO tether, already in place at the surface,was an obvious route to retention of structure, as seen in FIG. 4. Thethree traces in the FIG. 4 represent the thermograms for protein insolution, protein adsorbed onto bare PS particles, and proteins attachedto the surface through the Pluronic™ F108 intermediate. As withfibronectin, all cooperative transitions are absent from theprotein-particle adsorption complex. However, the PEO-tethered sampleshows the characteristic complex melting curve of native HSA, reflectingthe differential collapse of the three lobes of this protein. Thetransition enthalpies associated with thermal unfolding of HSA in thethree different states are listed in Table 2. Due to the irreversiblenature of these thermal transitions it should be noted that the listedvalues represent apparent enthalpies. TABLE 2 Human Serum Albumin T_(m)(° C.) ΔH(K cal/mol) In PBS 60-75 580 Conjugated to EGAP 60-75 460

Example 4 Cells Do Not Adhere to Hydrophobic Surface Coated with BlockCopolymer Surfactants

[0133] Osteoblast cells were seeded onto a polystyrene substrate whichhad been treated with C F108 or only in a localized circular area in thecenter of the substrate. As illustrated in FIG. 8, cells which wereseeded in serum containing media did not attach to the PEO modified areabut were found to attach, spread, and proliferate well on unmodifiedareas. FIG. 8a is a close-up displaying cells aligned at an interfacebetween PEO treated and unmodified areas. In FIG. 8b, cells were fixedand removed from culture well after adequate time to lay down asubstantial ECM. A dark spot in the center corresponds to the PEOtreated area where there were no cells.

Example 5 EGAP is Not Toxic to Primary Breast Epithelial Cells

[0134] Surgical discard from reduction mammoplasty was digested bystandard method into single and small cell aggregates of epithelialcells. Cells were placed on a Pluronic™ coated tissue culture plastic inCDM# media. Following a two week incubation at 37° C., 95% of cells werealive as indicated by the vital dyes.

Example 6 NIH 3T3 Cells Attach to and Grow on RGDS Conjugated EGAP

[0135] NIH 3T3 cells were attached to and grown on GRGDS conjugated EGAPas follows. EGAP formation and GRGDS conjugation was carried outessentially as described above. Briefly, the hydroxyl ends of Blockcopolymer surfactant was activated to form an EGAP using 4-nitrophenylchloroformate followed by 2-pyridyl disulfide.

[0136] A portion of the EGAP was derivatized with the Bolton-HunterReagent. This allows the polymer to be labeled with radioactive iodineand thus provides a means to accurately determine the surfaceconcentration of the EGAP.

[0137] GRGDS peptide was synthesized with a tyrosine residue at itscarboxyl terminus using methods well known in the art. The tyrosineresidue allows incorporation of radioactive iodine and thereby enablesaccurate determination of surface peptide concentration.

[0138] ESCA analyses of PS modified with F108 demonstrate that a highdegree of PEO coverage is obtained. As illustrated in Table 3 below,this result has been confirmed by contact angle measurements which showthat a substantial increase in the degree of substrate hydrophilicityoccurs upon coating PS with F108. TABLE 3 Substrate Average ContactAngle Untreated Polystyrene 82 F-108 Coated Polystyrene 68

[0139] The plateau concentration of F-108 adsorbed onto PS wasdetermined by isotope ¹²⁵I labeling and was found to be 3.3 mg/m². Thiscorresponds to one triblock every 7.4 nm². ESCA measurements have alsobeen used to confirm the presence of active sites for peptide couplingon PS coated with derivatized triblocks and the presence of peptides onPS conjugated with peptide via activated triblocks (Table 4). TABLE 4Substrate Treatment Element Atom % F108-PS Rxn w/AgNO₃ Ag 0.2PDS-F108-PS Rxn w/AgNO₃ Ag 0.8 PDS-F108-PS N 0.3 PDC-F108-PS Rxn w/GRGDSN 0.8

[0140] As a further means for characterizing PEO and GRGDS modified PSsubstrates, cell cultures were grown on conjugated EGAP coated culturesurfaces. NIH 3T3 fibroblasts were seeded onto GRGDS conjugated EGAPcoated polystyrene (RGD-PS), EGAP coated polystyrene (PDS-F108-PS), andF108 coated polystyrene (F108-PS) substrates at approximately 1×10⁴/cm².Thirty minutes after seeding, the substrates were gently washed. Theattached cells were incubated for 24 hours, after which, the substrateswere again gently washed and fixed for counting. As illustrated in FIG.9, RGD-PS were found to support cell adhesion, PDSF108-PS displayed anintermediate level of adhesiveness, and F108 coated polystyrene wasrelatively non-adhesive to fibroblast cells.

Example 7 NIH 3 T3 Cells Attach to GRGDSY Conjugated EGAP

[0141] Fibronectin peptide Gly-Arg-Gly-Asp-Ser-Tyr or GRGDSY wasconjugated to activated F108 (EGAP) and used to coat polystyrene (PS)culture dishes as described above. Cells were seeded at 6×103 cells/cm²in DMEM supplemented with 10% bovine serum. Substrates were washed after24 hrs. The attachment of NIT 3T3 cells to GRGDSY conjugated F108(PDSF108/RGD)was compared with untreated PS surface (PS), PS surfacecoated with F108 alone (F108), F108 adsorbed PS treated with GRGDSY (notconjugated) (F108/RGD), and pyridyl disulfate activated F108 (EGAP)adsorbed PS without GRGDSY (PDSF108).

[0142] The results of NIH 3T3 attachment to these various surfaces issummarized below. TABLE 5 Substrate Cell/cm² Standard Dev. of Mean F1080 0 F108/RGD 13 9.6 PDSF108 462 38.5 PDSF108/RGD 5331 465.4 PS 5821325.0

[0143]FIGS. 5 and 6, respectively, illustrate that NIH 3T3 cells doadhere and spread processes on GRGDSY conjugated EGAP surfaces, but donot adhere to F108 treated surface.

What is claimed and desired to be secured by United States LettersPatent is:
 1. A method for the attachment of organisms and molecules forgrowth or biological analysis comprising the steps of: a. contacting ahydrophobic surface with an EGAP for a time sufficient for said EGAP tobe adsorbed by the hydrophobic surface; b. conjugating a naturalor-recombinant biomolecule to the EGAP adsorbed to the hydrophobicsurface to form a biomolecule conjugated EGAP surface; c. contactingsaid biomolecule conjugated EGAP coated surface with at least oneorganism or molecule such that said organism or molecule adheres to thebiomolecule conjugated EGAP coated surface.
 2. The method according toclaim 1 wherein the biomolecule is selected from the group consisting ofnatural or recombinant proteins, peptides, amino acids, nucleic acids,lipids, and carbohydrates.
 3. The method according to claim 1 whereinthe biomolecule is selected from the group consisting of natural orrecombinant extracellular matrix proteins, adhesive proteins andcombinations thereof.
 4. The method according to claim 1 wherein thebiomolecule is selected from the group consisting of natural orrecombinant growth factor, mitogens, growth peptides, differentiatingfactors and combinations thereof.
 5. The method according to claim 1wherein the biomolecule is selected from the group consisting of naturalor synthetic sugars, carbohydrates, polysaccharides and combinationsthereof.
 6. The method according to claim 1 wherein the biomolecule isselected from the group consisting of a natural or synthetic lipids,sterols, fatty acids and combinations thereof.
 7. The method accordingto claim 1 wherein the EGAP is synthesized by reacting the blockcopolymer surfactant with 4-nitrophenylchloroformate followed by2-(2-pyridyldithio)ethylamine.
 8. The method according to claim 1wherein the biomolecule contains a thiol.
 9. The method according toclaim 1 wherein the biomolecule has been artificially thiolated.
 10. Themethod according to claim 8 wherein the biomolecule is coupled to theEGAP via a disulfide linkage.
 11. The method according to claim 8wherein the EGAP is formed from a block copolymer surfactant having theformula: (HO—PEO)_(c)(OH—PEO)_(d)(PPO)_(b) wherein b is an integer from1 to 3, (c+d) is an integer between 1 and 6, c is an integer between 0and 5, and d is at least 1, where PEO is of the formula:—(—C₂H₄—)—)_(u)— where u is greater than 50, where PPO is of theformula: —(—C₃H₆—O—)_(v)— where v is greater than
 25. 12. The methodaccording to claim 1 wherein the surface is contacted with an organism,and wherein said organism is a eukaryotic or prokaryotic cell.
 13. Themethod according to claim 1 wherein the surface is contacted withorganism, and wherein said organism is a virus.
 14. The method accordingto claim 1 wherein the surface is contacted with an molecule, andwherein said molecule is an antibody.
 15. The method according to claim1 wherein the surface is contacted with an molecule, and wherein saidmolecule is an pharmaceutical drug.
 16. A method for the attachment oforganisms and molecules to a surface for growth or biological analysiscomprising the steps of: a. modifying a block copolymer surfactant witha reactive group to obtain an EGAP; b. contacting a hydrophobic surfacewith said EGAP for a time sufficient for the EGAP to be adsorbed by thehydrophobic surface; c. conjugating a thiol containing biomolecule tosaid EGAP to form a biomolecule conjugated EGAP coated surface; d.contacting the biomolecule conjugated EGAP coated surface with anorganism or molecule such that said organism or molecule adheres to thebiomolecule conjugated EGAP coated surface.
 17. The method according toclaim 16 wherein the thiol containing biomolecule is selected from thegroup consisting of proteins, peptides, amino acids and combinationsthereof.
 18. The method according to claim 16 wherein the thiolcontaining biomolecule is an extracellular protein.
 19. The methodaccording to claim 16 wherein the thiol containing biomolecule is anadhesion protein.
 20. The method according to claim 16 wherein the thiolcontaining biomolecule is a growth factor.
 21. The method according toclaim 16 wherein the block copolymer surfactant is modified with areactive group selected from the group consisting of hydrozino,thiopyridyl, tyrosyl, malcimide, 2-pyridyl disulphide, 5-nitro-2-pyridyldisulphide, 4-pyridyl disulphide, 5-carboxy-2-pyiidyl disulphide, andthe nitrogen oxides of 2-pyridyl disulfide, 5-nitro-2-pyridyl disulfide,4-pyridyl disulfide, and 5-carboxy-2-pyidyl disulphide.
 22. The methodaccording to claim 16 wherein the biomolecule is artificially thiolated.23. The method according to claim 22 wherein the biomolecule is coupledto the EGAP via a disulfide linkage.
 24. The method according to claim16 wherein the EGAP is formed from a block copolymer surfactant having aformula: (HO—PEO)_(c)(OH—PEO)_(d)(PPO)_(b) wherein b is an integer from1 to 3, (c+d) is an integer between 1 and 6, c is an integer between 0and 5, and d is at least 1, where PEO is of the formula:—(—C₂H₄—)—)_(u)— where u is greater than 50, where PPO is of theformula: —(—C₃H₆—O—)_(v)— where v is greater than
 25. 25. The methodaccording to claim 16 wherein the surface is contacted with an organism,and wherein said organism is a eukaryotic or prokaryotic cell.
 26. Themethod according to claim 16 wherein the surface is contacted with anorganism, and wherein said organism is a virus.
 27. The method accordingto claim 16 wherein the surface is contacted with an molecule, andwherein said molecule is an antibody.
 28. The method according to claim16 wherein the surface is contacted with an molecule, and wherein saidmolecule is an pharmaceutical drug.
 29. A method of selecting at leastone desired organism or molecule from a mixture of at least twoorganisms or molecules comprising the steps of: a. contacting ahydrophobic surface with an EGAP for a time sufficient for said EGAP tobe adsorbed by the surface; b. conjugating a biomolecule to said surfaceattached EGAP to yield a biomolecule conjugated EGAP coated surface,said biomolecule being unique for a desired organism or molecule beingselected; c. contacting the biomolecule conjugated EGAP coated surfacewith a mixture of organisms or molecules containing the desired organismor molecule; d. allowing the desired organism or molecule to adhere tothe biomolecule conjugated EGAP coated hydrophobic surface; e. removingthe non-adhered organisms or molecules.
 30. The method according toclaim 29 wherein the EGAP is conjugated to the biomolecule before it isadsorbed onto the hydrophobic surface.
 31. The method according to claim29 wherein the desired organism or molecule is separated from undesiredorganisms or molecules by fluid dynamics.
 32. The method according toclaim 29 wherein the desired organism or molecule is separated fromundesired organisms or molecules by magnetic means.
 33. The methodaccording to claim 29 wherein the separation means comprisesstreptavidin.
 34. The method according to claim 29 wherein the organismis a eukaryotic or prokaryotic cell.
 35. The method according to claim29 wherein the organism is a virus.
 36. The method according to claim 29wherein the molecule is an antibody.
 37. The method according to claim29 wherein the molecule is a pharmaceutical drug.
 38. A method ofcoating a hydrophobic biomaterial for use in mammals comprising thesteps of: a. contacting an EGAP to a hydrophobic biomaterial for a timesufficient for said EGAP to be adsorbed by the biomaterial; b.conjugating a biomolecule to the EGAP to form a biomolecule conjugatedEGAP coated biomaterial; c. contacting the mammal with the biomoleculeconjugate EGAP coated hydrophobic biomaterial;
 39. The method accordingto claim 38 wherein the biomolecule is immunologically-inert such thatthe biomaterial does not induce an immune response in the mammal. 40.The method according to claim 39 wherein the immunologically-inertbiomolecule is hyaluronic acid.
 41. The method according to claim 39wherein the biomaterial is hydrophilic polymer coated with a hydrophobicmaterial.
 42. A biomolecule conjugated block copolymer surfactant havinga formula: (HO—PEO)_(c)(R—PEO)_(d)(PPO)_(b) wherein b is an integer from1 to 3, (c+d) is an integer between 1 and 6, c is an integer between 0and 5, and d is at least 1, where PEO is of the formula:—(—C₂H₄—)—)_(u)— where u is greater than 50, where PPO is of theformula: —(—C₃H₆—O—)_(v)— where v is greater than 25, and where R is abiomolecule selected from the group consisting of proteins, peptides,amino acids, nucleic acids, lipids, and carbohydrates.
 43. The compoundof claim 42 wherein the bond between R and PEO is a disulfide bond. 44.The compound of claim 42 wherein R is selected from the group consistingof natural or recombinant extracellular matrix proteins, growth factors,mitogens, growth peptides, and differentiating factors.
 45. The compoundaccording to claim 42 wherein R is selected from the group consisting ofnatural or synthetic sugars, carbohydrates, and polysaccharides.
 46. Thecompound of claim 41 wherein R is selected from the group consisting ofa natural or synthetic lipids, sterols, and fatty acids.
 47. Thecompound of claim 42 wherein R is a protein selected from the groupconsisting of a naturally thiol containing protein and artificiallythiolated protein.